Chitosan Matrix Dye Affinity Adsorbents For The Preparation And Purification Of The Protein Applied Research

Chitosan [poly- β (1â†' 4)-2-amino-2-deoxy-D-glucose] is produced by thedeacetylation of chitin, one of the most abundant polysaccharides. Due to a lot of reactive amino and hydroxyl groups on its molecules, Chitosan can easily be modified by chemical or physical processes to prepare varies functional sorbents, which have been widely used in medical, biomedical, food ingredients, printing and dyeing, cosmetics areas et al. Dye-affinity chromatography has become a routine step in protein purification, dye-ligand have been considered as one of the important alternatives to natural counterparts. In this paper, we prepared dye-affinity sorbents in which chitosan microspheres were used as the support, and two dye ligands (Cibacron Blue F3GA and Procion blue MX-R) were incorporated into these microbeads for separation and purification of proteins. The main work followes:1. Using epichlorohydrin as a crosslmking reagent, Chitosan microspheres were prepared by a inverse phrase cross-linking technique. The effect factors of preparation were studied in detail, and the favorable operation conditions were found at : the chitosan concentration is 3-5% , the proportion of oil to chitosan solution is 1-1.5:1,and the concentrations of epichlorohydrin and the porogen are 0.04mol/L and 0.5% respectively.The physical and chemical properties of the microspheres were characterized by using IR scan electron microscope. Results indicate that the microspheres prepared here have high porosity and low non-specific adsorption for proteins.2. Cibacron Blue F3GA and Procion blue MX-R were covalently coupled with Chitosan microspheres via the nucleophilic substitution reaction between the chloride of their triazine ring and the hydroxyl groups of the Chitosan molecules under alkaline conditions, and thus two novel dye-affinty sorbent CS-CBA and CS-PBR were obtained. The ligand density can be easily adjusted by the adding values. It was found that these dye-attached microspheres were stable in acidic and alkaline solution while the ligand leakage was very low.3. The affinity sorbent CS-CBA carrying 35.8 mg Cibacron Blue F3GA/g bead was used in the catalase adsorption studies. The maximum adsorption was observed at pH 7.0. The adsorption capacity decreased with an increase of ionic strengh. From adsorption isotherm at 25℃ which followed the Langmuir equation,the maximum adsorption capacity and dissociation constant were determined as respectively.The model of Chase was adopted to study the kinetic of adsorption, and we found the forward reaction velocity constant k1 was 0.036mL/mg.min.High desorption ratios (over 91% of the adsorbed catalase) were achieved by using l.0mol/L NaSCN (pH 8.0) . It appears that CS-CBA could be applied in adsorption and purification of catalase without significant denaturation.4. The affinity sorbent CS-PBR carrying Procion blue MX-R 8.35 mg /g bead was used in the human serum albumin adsorption studies. The maximum adsorption was observed at pH 5.0. The adsorption capacity decreased with an increase of ionic strengh. From adsorption isotherm at 25℃ which followed the Langmuir equation, the maximum adsorption capacity and dissociation constant were determined as respectively. The model of Chase was adopted to study the kinetic of adsorption, and we found the forward reaction velocity constant k\ was 0.029mL/mg.min. High desorption ratios (over 88% of the adsorbed albumin) were achieved by using 0.5 mol/L NaSCN (pH4.0).